Toxicology in Vitro 29 (2015) 544–550

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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit

In vitro lead exposure changes DNA methylation and expression of IGF2 and PEG1/MEST q Monica D. Nye a,b, Cathrine Hoyo c, Susan K. Murphy a,⇑ a

Duke University Medical Center, Department of Obstetrics and Gynecology, B225 LSRC, Research Drive, Durham, NC 27708, USA University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, 450 West Drive, Chapel Hill, NC 27599, USA c Department of Biological Sciences, North Carolina State University, Raleigh, NC 27695, USA b

a r t i c l e

i n f o

Article history: Received 4 August 2014 Accepted 6 January 2015 Available online 14 January 2015 Keywords: DNA methylation Epigenetics Insulin-like growth factor 2 (IGF2) Paternally expressed gene 1/mesoderm specific transcript (PEG1/MEST) Lead acetate Bisulfite pyrosequencing

a b s t r a c t Epigenetic processes, such as changes in DNA methylation, likely mediate the link between environmental exposures in utero and altered gene expression. Differentially methylated regions (DMRs) that regulate imprinted genes may be especially vulnerable to environmental exposures since imprinting is established and maintained largely through DNA methylation, resulting in expression from only one parental chromosome. We used the human embryonic kidney cell line, HEK-293, to investigate the effects of exposure to physiologically relevant doses of lead acetate (Pb) on the methylation status of nine imprinted gene DMRs. We assessed mean methylation after seventy-two hours of Pb exposure (0–25 lg/dL) using bisulfite pyrosequencing. The PEG1/MEST and IGF2 DMRs had maximum methylation decreases of 9.6% (20 lg/ dL; p < 0.005) and 3.8% (25 lg/dL; p < 0.005), respectively. Changes at the MEG3 DMRs had a maximum decrease in methylation of 2.9% (MEG3) and 1.8% (MEG3-IG) at 5 lg/dL Pb, but were not statistically significant. The H19, NNAT, PEG3, PLAGL1, and SGCE/PEG10 DMRs showed a less than 0.5% change in methylation, across the dose range used, and were deemed non-responsive to Pb in our model. Pb exposure below reportable/actionable levels increased expression of PEG1/MEST concomitant with decreased methylation. These results suggest that Pb exposure can stably alter the regulatory capacity of multiple imprinted DMRs. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The developmental origins of health and disease (DoHaD) hypothesis states that the origin of many diseases manifested later in life is established based on responses to perceived conditions in the in utero environment (Barker et al., 1989). Numerous epidemio-

Abbreviations: DMRs, differentially methylated regions; HEK-293, human embryonic kidney cells; Pb, lead (IV) acetate; H19, H19 imprinted maternally expressed transcript; IGF2, insulin-like growth factor 2; PEG1/MEST, paternally expressed gene 1/mesoderm-specific transcript; NNAT, neuronatin; PEG3, paternally expressed gene 3; PLAGL1, pleiomorphic adenoma gene-like 1; SGCE/PEG10, epsilon sarcoglycan/paternally expressed gene 10 (SGCE/PEG10); EDCs, endocrinedisrupting chemicals. q This work was supported by NIH Grants R25CA057726, R01DK085173, R01ES016772, P01ES022831 and by US EPA Grant RD-83543701. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the US EPA. ⇑ Corresponding author at: Department of Obstetrics and Gynecology, Duke University, Box 91012, Durham, NC 27708, USA. Tel.: +1 (919) 681 3423; fax: +1 (919) 684 5336. E-mail addresses: [email protected] (M.D. Nye), [email protected] (C. Hoyo), [email protected] (S.K. Murphy).

http://dx.doi.org/10.1016/j.tiv.2015.01.002 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.

logical studies support this hypothesis, for example, individuals exposed to severe caloric restriction in utero have a higher incidence of type 2 diabetes (Li et al., 2010; Ravelli et al., 1998) coronary heart disease (Roseboom et al., 2000), neurological disorders such as schizophrenia and ADHD (Kyle and Pichard, 2006), obesity (Ravelli et al., 1999), and breast cancer (Elias et al., 2004; Painter et al., 2006). Because of fetal programming, a stimulus or insult during sensitive periods of early life development can have permanent effects on structure, physiology and metabolism in later life (Godfrey and Barker, 2001; Lucas, 1994). Epigenetics plays a vital role in the ability to respond to nutritional and environmental factors that can lead to persistent changes in regulatory and growthrelated gene expression as an important component of fetal programming (Gluckman and Hanson, 2004; Santos and Dean, 2004). Epigenetic mechanisms, including modifications of histone proteins, DNA methylation, and the action of noncoding RNAs, all contribute to regulating repression or activation of gene expression. Due to its mitotic stability, DNA methylation is of particular interest with regard to determining if patterns of methylation can provide a record of past exposures (Heijmans et al., 2008, 2009; Hoyo et al., 2009; Mathers, 2007). The differentially methylated regions

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(DMRs) that regulate imprinted genes are established during gametogenesis and early embryogenesis and are stably maintained throughout somatic cell division (Hackett and Surani, 2013; Reik and Walter, 2001). These DMRs are reasonable for study as an archival record for several reasons. First, they have a known baseline level of methylation from which deviations are detectable; second, these regions are critical for the appropriate establishment and maintenance of imprinted gene expression; third, imprinted genes are essential for appropriate placental function, early development and growth; fourth, the DMRs exhibit parental origin-specific methylation profiles that are spatially and temporally stable such that methylation changes at imprinted gene DMRs resulting from in utero exposure to famine have been detected six decades postexposure (Heijmans et al., 2008); and last, these DMRs are known to exhibit shifts in methylation in response to a wide variety of other exposures, including maternal stress (Heijmans et al., 2008; Hochberg et al., 2011; Hoyo et al., 2011; Joubert et al., 2012; Murphy et al., 2012a; Timmermans et al., 2009; Tobi et al., 2009), and are widely deregulated in cancer (Sharma et al., 2010). The DMRs of many imprinted genes in humans have been characterized (Hoyo et al., 2011; Murphy et al., 2012b; Skaar et al., 2012; Woodfine et al., 2011) and provide relevant loci to investigate possible epigenetic responses resulting from environmental exposures (Coolen et al., 2011). In the US, 24 million households have Pb paint and increased levels of Pb-contaminated dust (CDC, 2012). Children are the most susceptible to Pb exposure and poisoning because they have an extended mouthing and teething phase of development, which can expose them to Pb-containing dust, the most common route of exposure. During childhood there are thought to be windows of vulnerability during which environmental exposure-mediated epigenetic changes can have detrimental consequences for adult health (Martorell et al., 2010; Sullivan, 2010; Victora et al., 2008) In the Cincinnati Lead Study, heightened vulnerability to Pb exposure occurs up to age 7 years, during which imprinted DMRs may become epigenetically deregulated.1 Those at greatest risk for Pb exposure are those children living at or below the poverty level, in older housing, and children who are members of ethnic minority groups (CDC, 2012). A growing body of research has shown that, even at low levels, Pb exposure has negative biological consequences. Exposure to 0.96). Pyrosequencing assay design used PSQ Assay design software (Qiagen; Valencia, CA). The percentage of methylation for each CpG in the target sequence was calculated using PyroQ CpG software (Qiagen). All pyrosequencing assays were performed in duplicate, and values shown represent the mean methylation of the CpG sites contained within the region analyzed. 2.3. qRT-PCR assay We used TaqMan Assays for PEG1/MEST (Hs00853380_g1), IGF2 (HS00171254) and beta-2-microglobulin (HS00187842) (Invitrogen, Carlsbad, CA). Using reverse transcriptase qPCR, cDNA was synthesized from 2 lg total RNA using oligo-dT priming and the SuperScript II Reverse Transcriptase first-strand synthesis kit (Invitrogen, Carlsbad, CA). The Applied Biosystems 7900H Fast Real Time PCR system (Foster City, CA) was used for real-time qRT-PCR. Relative PEG1/MEST and IGF2 expression levels were calculated using the delta delta Ct method with normalization to beta-2microglobulin. For relative gene expression values, each dose of Pb was normalized to the control and multiplied by 100 for graphical representation of the data. 2.4. Data analysis For each DMR, we measured methylation at multiple CpG sites: four for H19, three for IGF2, eight for MEG3, four for MEG3-IG, four for PEG1/MEST, three for NNAT, ten for PEG3, six for PLAGL1, and six for SGCE/PEG10. We then calculated the mean methylation for these CpG sites at each DMR. The mean DNA methylation fractions at the individual CGs were analyzed and compared among the different treatment groups. We have previously reported that Cronbach’s alphas for all DMRs were >0.89 allowing the use of mean methylation as the variable (Liu et al., 2012). Analysis of variance (ANOVA) was performed to determine the difference between groups for mean methylation followed by post-hoc Dunnett’s test for multiple comparisons. GraphPad Prism 6 statistical software was used to perform all analyses (GraphPad Prism, CA).

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these particular DMRs are only minimally malleable to Pb exposure in this cell type. Two DMRs, MEG3 and MEG3-IG, had methylation profiles that showed more variability in response to Pb exposure, but did not reach statistical significance (Fig. 1, bottom graphs). For the MEG3 DMR, the mean methylation decreased up to 2.1% with Pb exposure (Fig. 1, bottom left). MEG3-IG DMR had a maximum reduction of 1.8% (Fig. 1, bottom right). The IGF2 DMR showed a significant reduction in mean methylation in response to Pb treatment; the data after 72 h of Pb treatment are presented in Fig. 2. This decrease was most pronounced at 20 and 25 lg/dL relative to the control. A decrease of 4.3% (p < 0.001) was observed at 20 lg/dL, and at the highest dose, there was a 3.9% decrease in mean methylation (p < 0.001). We investigated the effects of Pb treatment on IGF2 gene expression using qPCR, and observed an overall decrease in expression over the full range of exposure. Between 0 lg/dL and 5 lg/dL, relative gene expression decreased by 7.5% but then increased 2% comparing the 5 lg/dL to the 10 lg/dL and 15 lg/dL doses. At 20 and 25 lg/ dL, relative expression was similar to that of the control. These data suggest that lower levels of Pb alter the expression of IGF2 more so than higher Pb doses. After Pb treatment, the methylation profile of PEG1/MEST showed the most dramatic shifts of all the DMRs in mean methylation

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Exposure of the human embryonic kidney cell line HEK-293 to physiologically relevant doses of lead acetate (Pb) were investigated to determine if these doses elicit changes in DNA methylation profiles for nine imprinted gene DMRs. We used bisulfite pyrosequencing to quantitatively measure the level of methylation at CpG sites within these regions, including DMRs that regulate the genes H19 Imprinted maternally expressed transcript (H19), Insulin-like Growth Factor 2 (IGF-2), Paternally Expressed Gene 1/Mesoderm-Specific Transcript (PEG1/MEST), Neuronatin (NNAT), Paternally Expressed Gene 3 (PEG3), Pleiomorphic Adenoma Gene-Like 1 (PLAGL1), Epsilon Sarcoglycan and Paternally Expressed Gene 10 (SGCE/PEG10), and two DMRs within the chromosome 14q32.2 imprinted domain: the Maternally Expressed Gene 3 (MEG3) promoter region (MEG3 DMR); and the DMR in the intergenic region between Delta-like 1 homolog (DLK1) and MEG3 (MEG3-IG DMR). There were five DMRs (H19, NNAT, PEG3, PLAGL1, and SGCE) that showed a 60.5% fluctuation in methylation levels across the full range of Pb exposures relative to the mock treated control; we classified them as non-Pb-responsive in our model (Fig. 1). Furthermore, Pb doses from 35 to 55 lg/dL also showed no significant change in methylation at these regions (data not shown), indicating

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Fig. 1. Mean methylation of the non-responsive to Pb DMRs: H19, NNAT, PEG3, PLAGL1, SGCE, MEG3-promoter, MEG3-IG, H19, NNAT, PEG3, PLAGL1, SGCE show little change in response to Pb treatment in HEK-293 cells. MEG3-promoter and MEG3-IG show slight change in response to Pb treatment. HEK-293 cells were treated for 72 h. Shown is the mean ± standard error for four independent experiments plated in triplicate (n = 12) for each dose of Pb.

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Fig. 2. IGF2 mean methylation and gene expression significantly altered. Mean methylation of the IGF2 DMR and relative IGF2 gene expression in response to 0–25 lg/dl Pb for 72 h. Shown is the mean methylation ± standard error for four independent experiments plated in triplicate, and for duplicate measures of IGF2 gene expression, which was normalized to B2M. Relative gene expression values were normalized to the control and multiplied by 100 for graphical representation of the data. ⁄ p < 0.01, ⁄⁄ p < 0.001.

Fig. 3. Mean methylation of PEG1/MEST is decreased with Pb treatment while PEG1/MEST gene expression is increased. Pb treatments, 0–25 lg/dL, for 72 h results in a significant decrease in mean methylation while gene expression is increasing. Shown is the mean methylation ± standard error for four independent experiments plated in triplicate, and duplicate measures of PEG1/MEST gene expression, which were normalized to B2M. Relative gene expression values were normalized to the control and multiplied by 100 for graphical representation of the data. ⁄ p < 0.005, ⁄⁄ p < 0.001, + marginal significance.

(Fig. 3). There was a reduction in mean methylation at all doses, from 61.5% in the control to 54.6% and 55.7% methylation at the two lowest doses. However, the most dramatic change was at the 20 lg/dL dose, where there was a 9.6% reduction in mean methylation (p < 0.0001). At the 25 lg/dL dose methylation was 55.7%. We next investigated the effects of Pb treatment on PEG1/MEST expression and found that expression increased at doses P10 lg/dL at the same time that methylation decreased in response to Pb treatment (Fig. 3). We observed the most prominent change in PEG1/MEST expression between 5 and 10 lg/dL Pb.

4. Discussion Our key findings are that doses of Pb below the actionable levels recommended by the CDC alter regulatory methylation with consequent changes in expression for two imprinted genes using a human cell culture model of Pb exposure. We found that the PEG1/MEST DMR was the most vulnerable to Pb exposure with a 5.8–9.6% reduction in mean methylation across the 0–25 lg/dL dose range. For the IGF2 DMR, Pb exposure reduced methylation up to 4.3% across the same dose range. We observed a trend towards decreased methylation for the MEG3 and MEG3-IG DMRs, although changes at these DMRs did not reach statistical significance. There was no change in mean methylation at the H19, NNAT, PEG3, PLAGL1, and SGCE/PEG10 DMRs and therefore we consider them non-responsive to Pb in this model. Overall these findings suggest that, at the actionable Pb levels for children and adults,

there are certain DMRs that are vulnerable to Pb treatment and others for which methylation levels are stably maintained within this cell model. The public health significance is that people exposed to even low levels of Pb may be more likely to have altered methylation at regions regulating imprinted genes if the results obtained here are also relevant in other cell types and to what occurs in vivo. Many imprinted genes are expressed in the brain and play a role in behavior and cognition in humans and animals (Davies et al., 2005; Isles and Wilkinson, 2000; Lefebvre et al., 1998). Epidemiological studies suggest that environmental exposures during development have a role in susceptibility to neurodevelopmental diseases and disorders in later life (Hou et al., 2012). Evidence from animal studies further supports the link between early environmental exposure and epigenetic shifts in conferring disease susceptibility (Hou et al., 2012). We investigated Pb as an environmental toxicant and showed that human cells exposed to this heavy metal exhibit functional methylation shifts at imprinted gene DMRs, including significant changes in mean methylation at the PEG1/MEST and IGF2 DMRs with consequent alterations in PEG1/MEST and IGF2 transcriptional activity. We observed non-monotonic dose responses for IGF2, MEG3, MEG3-IG, and SGCE/PEG10, where lower doses of Pb had a greater effect on DNA methylation (Figs. 1 and 2) and on IGF2 and PEG1/ MEST gene expression (Figs. 2 and 3). A common theme in toxicology is ‘dose makes the poison’, where it has been widely accepted that higher doses cause greater effects. As we have found here this may not always be the case. There are some biological responses

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that are more prominent at low doses of exposure, suggesting the need to further study low-level exposure to toxicants. In hormone receptor studies, endocrine-disrupting chemicals (EDCs) have been shown to produce non-monotonic dose response curves, and in the past 20 years several studies have identified EDCs that mimic or disrupt hormone function at low doses in ways not predicted by high-dose studies (Myers et al., 2009). Heavy metals have been classified as EDCs, so it is likely that the observed low dose effects on DNA methylation and gene expression in our study can be explained by this lack of a clear dose–response relationship (Schug et al., 2011). We hypothesize that, in addition to DNA methylation, histone modification and/or miRNA regulation may also contribute to the non-monotonic dose responses observed. PEG1/MEST is an imprinted gene located at chromosome 7q32.2 that encodes a member of the a/b-hydrolase fold family. In mice, disruption of Peg1/Mest expression causes growth retardation, defective maternal nurturing and increased pup lethality (Lefebvre et al., 1998). It is not currently known if PEG1/MEST has similar functions in humans. However, deregulated methylation at this DMR in humans has been associated with invasive breast cancer (Pedersen et al., 1999) and cervical cancer (Vidal et al., 2014). Paternally expressed IGF2 is the most studied genomically imprinted gene and is located at chromosome 11p15.5. It encodes a mitogenic growth factor critically important to early growth and development and is frequently overexpressed in many types of cancer. Loss of imprinting (activation of the normally silent maternal allele) has been shown to occur in many cancers, including colorectal (Cui et al., 2002), pancreatic, ovarian, and Wilms’ tumors (reviewed in (Feinberg, 2004)). In addition, IGF2 DMR methylation is altered in response to in utero exposures such as famine (Heijmans et al., 2008), folic acid intake (Hoyo et al., 2011), and exposure to tobacco smoke (Murphy et al., 2012a). Murphy et al. (2012a) measured IGF2 DMR methylation and expression in umbilical cord blood and found a 1% change in methylation correlates with a twofold change in transcription; this suggests that as little as a 1% decrease in methylation at this DMR can produce an expression change theoretically equivalent to that observed with complete loss of IGF2 imprinting. Epigenetic mechanisms are proposed to link early exposure to adult-onset disease. The lack of suitable epigenetic targets to ascertain periconceptional, prenatal, and early postnatal environmental exposures has limited the ability to infer causality in retrospective epidemiologic studies. We have previously shown stability of DNA methylation at imprinted gene DMRs across fetal tissues and between birth and one year of age (Murphy et al., 2012b). To our knowledge this is the first study to demonstrate a connection between alterations of an imprinted gene’s methylation profile and Pb exposure in an in vitro model. Although Pb levels as high as 25 lg/dL are no longer commonly detected, such levels were more common several decades ago. This may have led to permanent alterations in the epigenetic profile of imprinted genes in the exposed humans that make relevant the study of the effects of high versus low Pb exposure and more importantly, may have serious implications for the health of those historically exposed to such doses. In 1991, the Secretary of the Department of Health and Human Services called Pb the ‘‘number one environmental threat to the health of children in the United States’’ (EPA). Pb exposure is ubiquitous in numerous ways: through air, drinking water, food, contaminated soil, deteriorating paint, and household dust. High concentrations of airborne Pb particles in the home can come from Pb-contaminated dust from outdoor sources, including contaminated soil tracked inside and use of Pb in soldering and stainedglass making. The effects of high Pb exposure on fetuses and young children can be severe, and includes delays in physical and mental development, lower IQ levels, shortened attention spans, and

increased behavioral problems (Canfield et al., 2003a,b; Needleman, 1989). Our findings that threshold actionable levels of Pb exposure resulted in DNA methylation and gene expression changes at IGF2 and PEG1/MEST support the idea that environmental exposures can be reflected by altered regulation of imprinted genes. The stability of imprinted gene DMR methylation, along with critical roles in growth and development, make these genes and DMRs suitable for further study as biomarkers of, or intermediate endpoints for, early life environmental exposures. We used human embryonic kidney cells for all assays, cells that are derived from the mesodermal germ layer. The mesoderm also gives rise to blood cells, the circulatory and reproductive systems, muscles, connective tissues, adipose tissue, the peritoneum, cartilage, bone and blood vessels. Since 10% of Pb is excreted in urine through the action of the kidneys, employing a kidney cell line for these studies supports that our findings have direct biologic relevance. However, the molecular mechanisms or biological importance of Pb exposure changing the methylation status of these particular genes in kidney cells is not fully understood. The use of only one cell line is a limitation of this study, and future studies should be performed using other cell types. Other groups have reported that Pb exposure increases the risk of developing renal cell carcinoma (Boffetta et al., 2011) and IGF2 dysregulation has previously been implicated in this disease (Nonomura et al., 1997; Oda et al., 1998) as well as in Wilm’s tumor (Cui et al., 2001), another cancer of the kidney. In addition, PEG1/MEST is involved in maintenance of stem cell self-renewal in the kidney (Dekel et al., 2006). These reports highlight the importance of these particular genes to kidney function and relevance to disease. However, our limited focus on DNA methylation restricts our ability to fully understand how in vitro Pb exposure relates to altered gene expression via other epigenetic mechanisms. DNA methylation is often highly coordinated with chromatin remodeling and methyl-DNA binding factors, and thus it is likely that Pb might also alter histone modifications and/or the function on noncoding RNA molecules.

5. Conclusions In conclusion we modeled Pb exposure in a human cell culture system and found significant alterations of DNA methylation at the DMRs regulating PEG1/MEST and IGF2 and changes in gene expression. These findings suggest that Pb is able to alter the regulatory capacity of these imprinted DMRs. A better understanding of the molecular mechanisms involved in epigenetic profile shifts at imprinted gene DMRs following Pb exposure are needed for causal connections between environmental Pb exposure and phenotypic consequences to be established.

Authors’ contributions MDN participated in the design of the study, performed the experiments, and drafted the manuscript. CH conceived the idea, participated in the design of the study, and revised it critically for intellectual content. SKM conceived the idea, participated in the design and coordination of the study, and revised it critically for intellectual content. All authors read and approved the final manuscript.

Conflict of Interest The authors declare that there are no conflicts of interest.

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